Tumor radiosensitization using gene therapy

ABSTRACT

The invention provides a method of radiosensitizing a tumor in a subject by contacting the tumor with a cytokine or a nucleic acid molecule encoding a cytokine. The invention also provides a method of radiosensitizing a tumor in a subject by administering, at a site other than the tumor, a cell genetically modified to express a cytokine. The invention further provides a method of reducing the severity of a cancer in a subject by administering a cytokine at the site of the tumor or by immunizing the subject at a site other than the tumor with tumor cells genetically modified to express a cytokine, and treating the tumor with radiotherapy.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates generally to cancer therapy and,more particularly, to compositions and methods for sensitizing a cancerin a subject to radiation therapy.

[0003] 2. Background Information

[0004] Improved methods and novel agents for treating cancer haveresulted in increased survival time and survival rate for patients withvarious types of cancer. For example, improved surgical andradiotherapeutic procedures result in more effective removal oflocalized tumors. Surgical methods, however, can be limited due, forexample, to the location of a tumor or to dissemination of metastatictumor cells. Radiotherapy also can be limited by these factors, whichlimits the dose that can be administered. Tumors that are relativelyradioresistant will not be cured at such a dose.

[0005] Immunotherapeutic methods also are being examined as a means totreat a cancer by stimulating the patient's immune response against thecancer. In particular, the role of cytokines, which are cellular factorsthat can modulate an immune response, is an important factor to considerwhen planning an immunotherapeutic procedure. For example, expression ofa cytokine such as interleukin-2 (IL-2) can increase the proliferationof T cells, which are involved in the cellular immune response against acancer.

[0006] It is well known, however, that cytokine administrationfrequently is associated with toxic effects that limit the therapeuticvalue of these agents. For example, severe hypotension and edema limitthe dose and efficacy of intravenous and intralymphatic IL-2administration. In addition, flu-like symptoms or fatigue often areassociated with the administration of various cytokines. The toxicity ofsystemically administered lymphokines is not surprising as these agentsmediate local cellular interactions and normally are secreted only invery small quantities.

[0007] To circumvent the toxicity of systemic cytokine administration,an alternative approach involving cytokine gene transfer into tumorcells has produced anti-tumor immune responses in several animal tumormodels. In these studies, the expression of cytokines following cytokinegene transfer into tumor cells resulted in a reduction in tumorigenicityof the cytokine-secreting tumor cells when implanted into syngeneichosts. Reduction in tumorigenicity has been reported in studies using,for example, IL-2, interferon-γ or interleukin-4. In addition, thetreated animals often developed systemic anti-tumor immunity and wereprotected against subsequent tumor cell challenges with unmodified tumorcells.

[0008] Although a single treatment modality such as radiation therapy,chemotherapy, surgery or immunotherapy can result in improvement of apatient, superior results can be achieved when such modalities are usedin combination. In particular, treatment with a combination ofradiotherapy, which can be directed to a localized area containing atumor, and chemotherapy or immunotherapy, which provide a systemic modeof treatment, can be useful where dissemination of the disease hasoccurred or is likely to occur. Unfortunately, the therapeuticusefulness of radiation therapy can be limited where the tumor cells arerelatively radioresistant, since the does is limited by the tolerance ofnormal tissue in the radiation field. Thus, there exists a need tosensitize cancer tumors to the effects of radiotherapy so that it canmore effectively reduce the severity of a tumor in a patient. Thepresent invention satisfies this need and provides related advantages aswell.

SUMMARY OF THE INVENTION

[0009] The invention provides a method of radiosensitizing a tumor in asubject by contacting the tumor with a cytokine such as interleukin-3 orgranulocyte-macrophage colony stimulating factor or granulocyte colonystimulating factor. For example, the invention provides a method ofradiosensitizing a tumor in a subject by contacting the tumor withinterleukin-3. As an additional advantage, a method of the inventionprovides an enhanced systemic immune response in the subject against thecancer.

[0010] The invention also provides a method of radiosensitizing a tumorin a subject by administering, at a site other than the tumor, animmunizing composition containing a cytokine and a tumor antigen. Forexample, the invention provides a method of radiosensitizing a tumor ina subject by administering, at a site other than the tumor, tumor cellsthat have been genetically modified to express a cytokine.

[0011] The invention further provides a method of reducing the severityof a cancer in a subject by immunizing the subject, at a site other thanthe site of the tumor, with tumor cells genetically modified to expressa cytokine express and secrete a cytokine, then administering aradiotherapeutic dose of radiation.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention provides a method of radiosensitizing atumor in a subject by contacting the tumor with a cytokine such asinterleukin-3 (IL-3), granulocyte-macrophage colony stimulating factor(GM-CSF) or granulocyte colony stimulating factor (G-CSF). As usedherein, the term “contacting,” when used in reference to a cytokine anda tumor, means that the cytokine is present in the location of thetumor, particularly in the location of a localized tumor. As disclosedherein, a tumor can be contacted with a cytokine, for example, byinjecting a solution containing the cytokine into the region of thetumor, by administering a nucleic acid molecule encoding a cytokine intothe region of the tumor, wherein the nucleic acid molecule is taken upby cells in the tumor such that the cytokine is expressed, or byadministering a cell that has been genetically modified to express (andsecrete) the cytokine into the region of a tumor.

[0013] Since an additional advantage of a method of the invention isthat a systemic immune response against the cancer occurs in thesubject, a composition containing a cytokine or a nucleic acid moleculeencoding a cytokine is referred to herein generally as an “immunizingcomposition.” In addition, the term “immunizing” is used generally torefer to the administration of such an immunizing composition to asubject.

[0014] The invention also provides a method of radiosensitizing a tumorin a subject by administering, at a site other than the tumor, animmunizing composition comprising a cell genetically modified to expressa cytokine. For example, the invention provides a method ofradiosensitizing a tumor in a subject by administering to the subject animmunizing composition comprising tumor cells genetically modified toexpress a cytokine. Such an immunizing composition can be administeredat the site of the tumor, in which case the cytokine is IL-3 or GM-CSFor G-CSF, or can be administered at a site other than the tumor site, inwhich case the cytokine can be an interleukin, an interferon, a tumornecrosis factor or a colony stimulating factor, and preferably is IL-3or GM-CSF or G-CSF.

[0015] In one embodiment of the invention, an immunizing composition,which contains a cytokine such as IL-3 or GM-CSF or G-CSF, isadministered at the site of a tumor in a subject. As a result, themicroenvironment of the tumor is altered such that a systemic immuneresponse against the tumor in the tumor occurs in the subject and thetumor is radiosensitized.

[0016] In another embodiment of the invention, an immunizingcomposition, which contains a cytokine and a tumor antigen, isadministered at a site other than the tumor site in the subject. As aresult of the administration of the immunizing composition at a siteother than a tumor site, a systemic immune response is stimulated in thesubject and the tumor can become radiosensitized.

[0017] An immunizing composition, which contains a cytokine or a nucleicacid molecule encoding a cytokine, can be administered to a subjecthaving a cancer. As a result, an immune response is stimulated in thesubject against the cancer, wherein the systemic immune response oralterations induced by the immune response in the tumor microenvironmentcan radiosensitize the subject's cancer. Subsequent radiotherapy thencan be used to treat the radiosensitized tumor and the systemic immuneresponse can destroy remaining tumor cells, including any metastaticlesions. Thus, in another embodiment, the invention provides a method ofreducing the severity of a cancer in a subject, comprising immunizingthe subject with an immunizing composition and administering a dose ofradiation to the site of the cancer.

[0018] As disclosed herein, immunization of a subject provides a meansto radiosensitize a tumor such that it can be treated more effectivelyby radiotherapy (see Example I). As used herein, the term “tumor” meansa localized growth of cancer cells, which can be the site where a canceroriginally formed or can be a metastatic lesion. The terms “tumor cell”and “cancer cell” are used interchangeably herein to mean a malignantcell.

[0019] A method of the invention is particularly useful for treating asubject having metastatic lesions that have disseminated from anoriginal tumor site because, in addition to radiosensitizing the tumor,a method of the invention also provides a systemic immune response,which can kill disseminated cancer cells. Thus, the invention is usefulfor treating a subject with a cancer such as a melanoma or any othercancer in which the dissemination of metastatic lesions is common, andprovides the additional advantage that recurrence of a tumor is lesslikely to occur following treatment.

[0020] A method of the invention radiosensitizes a tumor in a subject.As used herein, the term “radiosensitize,” when used in reference to atumor or a tumor cell, means to increase susceptibility of the tumor ortumor cell to the effects of radiation. It is recognized that the term“radiosensitize” is used in a comparative sense and, with regard to thepresent invention, indicates that the radiation dose to reduce theseverity of a cancer in a subject that has been immunized as disclosedherein is less than the radiation dose that would have been required ifthe subject had not been immunized. In contrast, the term“radioresistant” means that a cell is relatively refractory to theeffects of radiation.

[0021] As used herein, the term “effects of radiation” refers to thewell known cytostatic and cytotoxic effects that radiation has on acell. For example, exposure of a cell to radiation can inhibitprogression of the cell through the cell cycle; can damage nucleicacids, proteins, or other macromolecules in a cell; or can kill the cellby inducing apoptosis. It should be recognized that these effects ofradiation are interrelated and represent a continuum of effects, themagnitude of which is dependent, in part, on the radiation dose and onthe relative radiosensitivity of the target cell.

[0022] The present invention provides a means to radiosensitize a tumorin a subject, such that the tumor is more susceptible the effects ofradiation, including tumor cell killing. For convenience, reference ismade generally herein to tumor cell “killing.” It should be recognized,however, that an increased susceptibility of a tumor cell to any of theeffects of radiation can provide a significant therapeutic advantage toa cancer patient.

[0023] The effectiveness of a method of the invention in treating asubject can be identified using well known methods. For example, theeffectiveness of treatment can be identified by detecting, in a subjectimmunized as disclosed herein, prolonged survival of the subject,disappearance of the tumor, or a decreased rate of growth of anirradiated tumor as compared to the rate of growth prior to irradiation.In human cancer patients, such measurements generally are made usingwell known imaging methods such as magnetic resonance imaging,computerized axial tomography and X-rays. In addition, determination ofthe level of a tumor marker such as the detection of levels ofcirculating carcinoembryonic antigen (CEA) or prostate specific antigenor the like also can be used as an indication of the effectiveness of atreatment. Thus, the effectiveness of a method of the invention can bedetermined by measuring a decrease in the growth rate of a tumor or anappropriate change in the level of a circulating marker, the presence orrelative level of which is indicative of cancer.

[0024] Radiation therapy is a conventional method for treating cancer.In particular, radiotherapy is useful in cases where the tumor isrelatively localized and not excessively large, or where surgicalexcision of the tumor is contraindicated due, for example, to thelocation of the tumor. Radiation therapy is a preferred method oftreating, for example, prostate cancer and brain tumors. The skilledartisan would know the appropriate dosages, treatment schedules andradiation sources to use for treating a particular cancer.

[0025] Various factors can limit the usefulness of radiotherapy.Ultimately, however, the success of radiotherapy is limited due tounacceptable patient morbidity that occurs as a result of consequentirradiation of normal tissue in the radiation field. In particular,exposure of rapidly renewing tissues, including, bone marrow, smallintestine and skin, to radiation can lead to unacceptable patientmorbidity. However, slowly proliferating tissues, including nervoustissue, also can be damaged irreversibly if exposed to an excessivelyhigh dose of radiation.

[0026] Administration of radiotherapy as fractionated doses over aperiod of time can provide advantages over administration of a singlelarge dose. In particular, fractionated doses of radiation are useful ifthe cells in the normal tissue in the radiation field can repairradiation induced damage faster or more efficiently than the tumor cellsin the radiation field. In this case, fractionated doses can beadministered at intervals that preferentially allow repair of the normalcells as compared to the tumor cells. In addition, tumors generally haverelatively hypoxic regions that are less susceptible to radiationdamage. Fractionated radiation doses also can permit reoxygenation tooccur in such regions, due to sloughing off of tumor cells killed byprevious doses, thus improving the effectiveness of subsequent radiationdoses.

[0027] Considerable research has been directed to the identification ofchemical agents that selectively increase the radiosensitivity of tumorcells, but not of normal cells. Such radiosensitizers can work, forexample, by effecting reoxygenation of a hypoxic region of a tumor or byacting as an oxygen mimetic. Since normal tissue is well oxygenated,such a radiosensitizer can increase the sensitivity of the tumor cells,while having relatively less effect on the normal cells, thuseffectively radiosensitizing the cancer cells.

[0028] Cytokines are a class of molecules that, in some cases, also canact as radiosensitizing agents. Cytokines constitute a family ofpolypeptides that are produced by leukocytes and other cells andregulate the immune and inflammatory responses in humans (Thomson, TheCytokine Handbook (Academic Press; 1994), which is incorporated hereinby reference; see Chap. 1). Cytokines are polypeptides or glycoproteins,including the heterodimeric IL-12, that bind to high affinity cellsurface receptors. Numerous cytokines, including interleukins 1-13(IL-1, IL-2, IL-3, etc.), interferons α, β and γ (Ifn-α, Ifn-β andIfn-γ), tumor necrosis factors α and β (TNF-α and TNF-β), colonystimulating factors such as macrophage colony stimulating factor(M-CSF), G-CSF and GM-CSF, and transforming growth factor β (TGFβ) andstem cell factor, are known in the art (Thomson, supra, 1994).

[0029] Cytokines can increase or decrease the rate of cell proliferationand can affect the differentiation state of a target cell, includingcells involved in the immune response. For example, IL-2, IL-4 and IL-7are T cell growth factors, which increase proliferation of T cells;M-CSF, GM-CSF, G-CSF and IL-3 can induce bone marrow cells todifferentiate into macrophages or granulocytes or both; and Ifn-γ, IL-4,IL-7 and GM-CSF can activate macrophages, increasing their tumoricidalactivity.

[0030] In addition, the expression of specific combinations of cytokinescan be particularly useful for stimulating an immune response. Forexample, expression of IL-1, IL-6 or a TNF can enhance IL-2 induced Tcell proliferation; IL-6 can enhance IL-4 induced T cell proliferation;and of Ifn-γ, IL-2 and IL-12 can stimulate T cells of the T helper-1class, which are involved in the cellular immune response. Thus, it canbe particularly useful to express specific combinations of cytokines forthe purpose of stimulating an immune response.

[0031] It is recognized, however, that the expression of othercombinations of cytokines can inhibit an immune response. For example,TGF-β can inhibit IL-2 action and IL-10 can inhibit cytokine productionand antigen-specific proliferation of T helper-1 cells. In addition,various types of cancer cells can express receptors for a cytokine suchas IL-2 or IL-1, such that expression of the cytokine can induceproliferation of the tumor cells. Thus, the selection of a cytokine orcombination of cytokines to administered to a subject must be consideredcarefully (Thomson, supra, 1989; see Chap. 25).

[0032] In some cases, cytokines can act to radiosensitize cancer cells.For example, tumor cells genetically modified to express IL-6 or IL-7were more sensitive to radiation induced killing in vitro than were thecorresponding unmodified tumor cells (McBride et al., Acta Oncol.34:447-451 (1995), which is incorporated herein by reference).Surprisingly, however, when unmodified tumor cells or IL-7 expressingtumor cells were injected into a mouse thigh, then treated withradiotherapy, the IL-7 expressing tumor cells were more radioresistantthan the unmodified tumor cells.

[0033] In other studies, human-tumor cells that were geneticallymodified to express TNF-A when irradiated, then xenografted into nudemice, were more sensitive to radiation therapy than were thecorresponding unmodified tumor cells (Weichselbaum et al., Canc. Res.54:4266-4269 (1994); Hallahan et al., Nat. Med. 1:786-791 (1995), eachof which is incorporated herein by reference). TNF-α also can act as aradioprotector, however, decreasing, for example, the lethal effect ofradiation in mice (Neta et al., J. Exp. Med. 175:689-694 (1992)). IL-1also has been described as providing a radioprotective effect (Neta etal., supra, 1992), although studies using another system found no suchradioprotective effect for IL-1, or for IL-2 or IL-3 (Gallicchio et al.,J. Biol. Resp. Mod. 8:479-487 (1989)).

[0034] As disclosed herein, cytokines such as IL-3, GM-CSF and G-CSF canbe useful to radiosensitize a tumor in a subject, and have theadditional advantage of stimulating a systemic immune response in thesubject. It is well known, however, that administration of a cytokine toa subject is limited by the generalized toxicity induced when the agentis administered systemically. Thus, cytokines may be better administeredlocally, for example, at the site of a tumor.

[0035] A cytokine can be administered by injection as a solution intothe site of a tumor. Furthermore, the cytokine can be administered as acytokine polypeptide, or can be administered in the form of a nucleicacid molecule encoding the cytokine, wherein the nucleic acid moleculeis taken up by a cell in the tumor and the cytokine is expressedtherefrom. Where a nucleic molecule encoding a cytokine is administeredto the site of a tumor, the nucleic acid molecule generally is linked toan appropriate regulatory such as a promotor that provides selectiveexpression of the cytokine at the site of the tumor (see, for example,Seung et al., Canc. Res. 55:5561-5565 (1995), which is incorporatedherein by reference). In addition, the nucleic acid molecule encodingthe cytokine can be linked to a vector such as a retrovirus vector (seebelow) or the nucleic acid molecule encoding the cytokine can bephysically associated with a formulation such as a liposome or an inertparticle such as gold.

[0036] Preferably, the cytokine is expressed from a cell such as a tumorcell that has been genetically modified, in vitro or in vivo, to expressthe cytokine. Expression of a cytokine from a genetically modified cellprovides the advantage that sustained, localized expression of thecytokine can occur, thus obviating the need for repeatedadministrations.

[0037] Various studies have been performed with tumor cells geneticallymodified to express a cytokine. As discussed above, for example, tumorcells that were genetically modified to express TNF-α and injected intounmodified tumors growing in immunodeficient mice, sensitized the tumorto radiation therapy more than did the corresponding unmodified tumorcells (Weichselbaum et al., supra, 1994; Hallahan et al., supra, 1995).In addition, human renal carcinoma cells that were genetically modifiedto express IL-2, Ifn-α, or both cytokines, then irradiated with a doseof radiation that inhibited the growth, but not cytokine expression, ofthe genetically modified cells, lost their tumorigenicity as determinedfollowing injection into T cell depleted mice (Belldegrun et al., J.Natl. Canc. Inst. 85:207-216 (1993), which is incorporated herein byreference). In addition, the genetically modified renal carcinoma cellsprevented the growth of unmodified renal carcinoma cells when injectedtogether, but not if the genetically modified cells were injected at adifferent site from the unmodified cells (Id.). These studies indicatethat tumor cells that are genetically modified to express a cytokine canbe used for local delivery of the cytokine to a desired site such as atumor.

[0038] In other studies, intraperitoneal immunization of mice witheither immunogenic and non-immunogenic tumor cells, each of which wasgenetically modified to express IL-3, protected the mice from laterchallenge with the corresponding unmodified tumor cells (McBride et al.,Folia Biolog. 40:62-73 (1994), which is incorporated herein byreference; see Example I). These results demonstrate that immunizationof IL-3 expressing tumor cells can stimulate a systemic immune responsethat protects against a later challenge with tumor cells.

[0039] As disclosed herein, the expression of a cytokine such as IL-3 orGM-CSF or G-CSF at the site of a tumor can radiosensitize the tumor in asubject and stimulates systemic immunity in the subject against thecancer. For example, a non-immunogenic murine fibrosarcoma tumor cellline, FSAN, or a moderately immunogenic murine fibrosarcoma tumor cellline, FSA (also called FSAR), was genetically modified by transductionusing a retroviral vector containing an expressible IL-3 encodingnucleic acid. FSAN cells that were genetically modified to express IL-3or genetically modified with a vector lacking the IL-3 coding sequencewere injected into mice, then, after the tumors attained a size of about6 to 8 mm, the tumors were irradiated with a single dose of 25 Gray(Gy), 40 Gy or 55 Gy of X-rays (see Example I). In all cases, the IL-3expressing tumors completely regressed after irradiation, whereasunirradiated IL-3 expressing tumors or irradiated tumor geneticallymodified with the control vector continued to grow. Furthermore,systemic immunity was stimulated in the cured mice, such that no tumorsdeveloped when the mice were subsequently challenged with unmodifiedtumor cells.

[0040] The results of Example I demonstrate that expression of IL-3 atthe site of tumor can radiosensitize a tumor and can induce a systemicimmune response in the subject against the cancer. In addition, whereadministration of an immunizing composition is at the site of tumor, acytokine such as GM-CSF or G-CSF also can be useful in the presentinvention. For example, the heterodimeric receptors for IL-3 and GM-CSFshare a common chain (Thomson, supra, 1994; see Chaps. 5 and 19),indicating that these cytokines can act, in part, through a sharedpathway such as through the stimulation of dendritic cells, which canincrease tumor antigen presentation. In addition, G-CSF can inducegranulocyte infiltration, as was observed in the IL-3 expressing tumorcells (Example I). Thus, administration of IL-3 or GM-CSF or G-CSF atthe site of a tumor can be useful to radiosensitive the tumor and tostimulate a systemic immune response against the cancer.

[0041] Where a tumor is radiosensitized by administering IL-3 or GM-CSFor G-CSF at the site of the tumor in a subject, it is not a necessaryrequirement to include a tumor antigen in the immunizing composition,since the tumor can provide the antigen. However, a cytokine, includingan interleukin, interferon, tumor necrosis factor or colony stimulatingfactor, also can be administered at site other than the tumor site andcan induce an immune response that can radiosensitize the tumor in thetumor. In this embodiment of the invention, the immunizing compositionalso can contain a tumor antigen in addition to the cytokine. Ifdesired, an immunizing composition also can contain an adjuvant such asBCG (see Harlow and Lane, Antibodies: a laboratory manual (Cold SpringHarbor Laboratory Press 1988); Mishell and Shiigi, Selected Methods inCellular Immunology (W. H. Freeman and Co. (1980)), each of which isincorporated herein by reference) or other adjuvant as commerciallyavailable (Ribi Immunochem Res., Inc.; Hamilton Mont.). As used herein,the term “immunizing composition” is used broadly to mean a cytokinethat is in a form for administration to a subject and, in addition, cancontain a tumor antigen or other immunostimulatory agent as desired.

[0042] When a cytokine is administered at a site other than the tumorsite, the cytokine is administered in a form that results in controlledrelease of desirably low levels of the cytokine. Thus, an immunizingcomposition can be formulated to contain a cytokine in combination witha material such as DepoFoam™, a wafer, an immunobead, a micropump orother material that provides for controlled slow release of thecytokine. Such controlled release materials are well known in the artand available from commercial sources (Alza Corp., Palo Alto Calif.;Depotech, La Jolla Calif.; see, also, Pardoll, Ann. Rev. Immunol.13:399-415 (1995), which is incorporated herein by reference).

[0043] In addition, a cytokine can be administered in combination with atumor antigen, which can be in the form of a tumor cell, a tumor cellextract or a purified tumor antigen. A tumor antigen can be obtainedfrom the subject or can be a known tumor antigen, including, forexample, epithelial cell mucin, which is encoded by the MUC-1 gene, orthe melanoma antigen, MZ2-E, which is encoded by the MAGE-1 gene, eachof which is associated with particular tumor cells (Finn, Curr. Opin.Immunol. 5:701-708 (1993), which is incorporated herein by reference).

[0044] An immunizing composition also can comprise a tumor cell, whichcan be obtained from the subject to be treated, that is geneticallymodified to express a cytokine. It should be recognized that, whilereference is made to the “expression” of a cytokine by a cell, such acell that is useful in the invention also must secrete the cytokine. Thegenetic modification of a subject's tumor cells to express a cytokineprovides the advantage that, in addition to expression of the cytokine,the tumor cell also presents a tumor antigen, against which an activeimmune response can be generated.

[0045] If a subject's tumor cells are not readily available, anothertype of cell can be genetically modified to express a cytokine and, inaddition, can be genetically modified to express a tumor antigen, whichcan be the same as the tumor antigen expressed on the subject's cancercells or can be a known tumor antigen as disclosed above. Suchgenetically modified cells, which express a tumor antigen and acytokine, are referred to herein as “carrier” cells (see PCT/US92/08999,filed Oct. 23, 1992, which is incorporated herein by reference).

[0046] Genetically modifying a cell to express a known tumor antigen canbe particularly useful when the tumor cells to be genetically modifiedare not obtained from the subject to be treated. For example, it may notbe possible to obtain a sufficient number of tumor cells from a cancerpatient or the patient's tumor cells may not be adaptable to growth inculture. In this case, cells that do not express a particular tumorantigen that is expressed by the patient's cancer cells can begenetically modified to express the tumor antigen and, in addition, canbe genetically modified to express a cytokine. Upon administration ofsuch a genetically modified carrier cell to the subject, the subject'simmune response against the cancer can be stimulated and a tumor in thesubject can be sensitized to radiation therapy.

[0047] A cytokine also can be expressed from a genetically modified cellsuch as a fibroblast or an antigen presenting cell such as a monocyte, adendritic cell or a lymphocyte. Such cells, which can be obtained fromthe subject or can be allogenic cells, are referred to herein as“cytokine-expressing cells” or “CE cells” and can be prepared asdisclosed herein or using methods well known in the art (seePCT/US92/08999, supra, 1992). Such CE cells can be administered at thesite of a tumor or, if administered at a site other than a tumor site,preferably, are administered in combination with a tumor antigen.

[0048] A cell such as a tumor cell that is genetically modified toexpress a cytokine can be further modified to express animmunostimulatory agent such as the costimulatory B7 molecules, B7.1 andB7.2, (Baskar et al., Proc. Natl. Acad. Sci, USA 90:5687-5690 (1993);Townsend and Allison, Science 259:368-370 (1993); Tan et al., J.Immunol. 149:32217-3224 (1992), each which is incorporated herein byreference), autologous MHC class I and class II molecules (Plautz etal., Proc. Natl. Acad. Sci., USA 90:4645-4649 (1993); Hui et al., FemsMicrobiol. Immunol. 2:215-221 (1990); Ostrand-Rosenberg et al., J.Immunol. 144:4068-4071 (1990), each of which is incorporated herein byreference), allogeneic histocompatability antigens such as HLA-B7 (Nabelet al., Proc. Natl. Acad. Sci., USA 90:11307-11311 (1993), which isincorporated herein by reference) and known tumor antigens (Finn, supra,1993). For example, a subject's cancer cell may not express an MHC classI or II molecule and, as a result, would not induce an optimal immuneresponse. In such a case, expression of the appropriate MHC molecule canbe useful for stimulating an immune response in the subject against thecancer. Methods for determining whether a tumor cell expresses aparticular immunostimulatory agent are known in the art and can be usedto determine whether the tumor cell should be genetically modified toexpress the immunostimulatory agent.

[0049] In some aspects of the invention, the administration of viabletumor cells is required. However, administration of viable tumor cellsto a subject requires that the tumor cells be inactivated so they do notgrow in the subject. Inactivation can be accomplished by any of variousmethods, including, for example, by irradiation, which is administeredat a dose that inhibits the ability of the cells to replicate but doesnot immediately kill the tumor cells. Where the irradiated tumor cell isa carrier cell that has been genetically modified to express a cytokine,it is known that such irradiation does not substantially affect theexpression of the cytokine (Belldegrun et al., supra, 1993). Treatmentof tumor cells with a cytostatic agent or with a low dose of a cytotoxicagent also can render the cells reproductively inactive. Such viabletumor cells can present tumor antigens to the subject's immune systembut cannot multiply and form new tumors.

[0050] It is further recognized that, in some cases, a tumor cell canexpress an immunosuppressive agent such as a TGFβ. Thus, if it isdesirable to use such a tumor cell as an antigen or as a carrier cell inthe present invention, the tumor cell can be genetically modified toreduce the expression of the immunosuppressive factor. Tumor cells thatproduce immunosuppressive factors are known in the art and are present,for example, in carcinomas, sarcomas, gliomas, melanomas, lymphomas andleukemias (Sulitzeanu, Adv. Canc. Res. 60:247-267 (1993), which isincorporated herein by reference). Whether a cancer cell is producing animmunosuppressive agent can be readily determined using methods asdisclosed herein or otherwise known in the art.

[0051] Immunosuppressive agents are known in the art and include, forexample, TGFβ, lymphocyte blastogenesis inhibitory factor, theretroviral p15E protein, suppressive E-receptor (see Sulitzeanu, supra,1993) and extracellular matrix molecules such as fibronectin andtenascin (Olt et al., Cancer 70:2137-2142 (1992); Hemasath et al., J.Immunol. 152:5199-5207 (1994), each of which is incorporated herein byreference). It is recognized, for example, that various isoforms of TGFβsuch as TGFβ1, TGFβ2, TGFβ3, TGFβ4 and TGFβ5 exist (see, for example,Roszman et al., Immunol. Today, 12:370-274 (1991); Constam et al., J.Immunol., 148:1404-1410 (1992); Elliot et al., J. Neuro-Oncology, 14:1-7(1992), each of which is incorporated herein by reference) and that theimmunosuppressive effect of one or more of these isoforms of TGFβdepends, for example, on the target cell. The term “TGFβ” is usedgenerally herein to mean any isoform of TGFβ, provided the isoform hasimmunosuppressive activity.

[0052] The present invention provides a method of reducing the severityof a cancer in a subject by administering to the subject an immunizingcomposition, which stimulates an immune response that radiosensitizes atumor in the subject, then administering a radiotherapeutic dose ofradiation to the tumor. As used herein, the term “reducing the severityof a cancer” means that the clinical signs or symptoms of the cancer ina subject are indicative of a beneficial effect to the subject due totreatment using a method of the invention.

[0053] Although complete remission is the optimal result, it isrecognized that any decrease in the rate of progression of the cancercan provide a palliative effect in the subject, thus improving thesubject's quality of life. Methods for determining whether a treatmentis reducing the severity of a cancer are well known in the art andinclude, for example, imaging methods such as magnetic resonanceimaging, computerized axial tomography and X-rays, and tumor markerassays such as the detection of levels of circulating carcinoembryonicantigen (CEA) or prostate specific antigen or the like or by detectingthe activation of immunoeffector functions in a subject such as theactivation of tumor cytolytic immunoeffector cells.

[0054] A method of the invention can reduce the severity of a cancer ina subject, by sensitizing a tumor in the subject to radiotherapy. Inaddition, a method of the invention can provide systemic immunityagainst the subject's cancer, such that cancer cells that are not killedby the radiotherapy are killed by the subject's immune response. Thepresence of such an immune response can be identified by comparing theimmune functions of a subject prior to administration of an immunizingcomposition with the immune functions following administration. Suchimmune functions can be determined using methods well known in the artfor measuring a humoral or cellular immune response (see, for example,Harlow and Lane, supra, 1988).

[0055] Genetic modification of a tumor cell or a fibroblast or antigenpresenting cell for use in the present invention is advantageous becausethe genetically modified cell provides sustained expression of thecytokine. Viral vectors such as retrovirus, adenovirus oradenovirus-associated vectors can be particularly useful for geneticallymodifying a cell (see, for example, Flotte, J. Bioenerg. Biomemb.,25:37-42 (1993) and Kirshenbaum et al., J. Clin. Invest, 92:381-387(1993), each of which is incorporated herein by reference; see, also,Hallahan et al., supra, 1995). Such vectors are particularly useful whenthe vector contains a promoter sequence, which can provide constitutiveor, if desired, inducible or tumor selective expression of a clonednucleic acid sequence. Such vectors are well known in the art (see, forexample, Meth. Enzymol., Vol. 185, D. V. Goeddel, ed. (Academic Press,Inc., 1990), which is incorporated herein by reference) and availablefrom commercial sources (Promega, Madison, Wis.). In particular, avector containing a radiation inducible promotor can be useful in thepresent invention (see, for example, Hallahan et al., supra, 1995).

[0056] Vectors can be introduced into a cell or into cells within atumor by any of a variety of methods known in the art (see, for example,Sambrook et al., Molecular Cloninq: A laboratory manual (Cold SpringHarbor Laboratory Press 1989); and Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1994), each ofwhich is incorporated herein by reference). Such methods include, forexample, transfection, lipofection, electroporation and infection withrecombinant vectors or the use of liposomes.

[0057] Introduction of nucleic acids by infection (transduction) using aviral vector is particularly advantageous in that it can be effective invitro or in vivo. Higher efficiency can also be obtained due to theinfectious nature of a viral vector. Moreover, viruses are veryspecialized and typically infect and propagate in specific cell types.Thus, their natural specificity can be used to target the vectors tospecific tumor cell types in a biopsy culture, which may be contaminatedwith other cell types. Viral or non-viral vectors can also be modifiedwith specific receptors or ligands to alter target specificity throughreceptor mediated events.

[0058] A nucleic acid molecule also can be introduced into a cell usingmethods that do not require the initial introduction of the nucleic acidsequence into a vector. For example, a nucleic acid sequence encoding acytokine can be introduced into a cell using a cationic liposomepreparation (Morishita et al., J. Clin. Invest., 91:2580-2585 (1993),which is incorporated herein by reference; see, also, Nabel et al.,supra, 1993)). In addition, a nucleic acid sequence can be introducedinto a cell using, for example, adenovirus-polylysine DNA complexes(see, for example, Michael et al., J. Biol. Chem., 268:6866-6869 (1993),which is incorporated herein by reference). Other methods of introducinga nucleic acid sequence are well known (see Goeddel, supra, 1990).

[0059] Nucleic acid sequences encoding various cytokines have beencloned and are available for use (GenBank; Thomson, supra, 1994).Nucleic acid sequences encoding, for example, cytokines such as variousinterleukins, including IL-3, interferons and colony stimulatingfactors, including GM-CSF and G-CSF, are available from the AmericanType Culture Collection (see ATCC/NIH Repository Catalogue of Human andMouse DNA Probes and Libraries, 6th ed., 1992) or are availablecommercially (Amgen, Thousand Oaks, Calif.; see, also, Patchen et al.,Exptl. Hematol., 21:338-344 (1993); Broudy et al., Blood, 82:436-444(1993), each of which is incorporated herein by reference).

[0060] Selectable marker genes encoding, for example, a polypeptideconferring neomycin (neo) resistance also are readily available and,when linked to a nucleic acid sequence or incorporated into a vector,allow for the selection of cells that have successfully incorporated thedesired nucleic acid sequence. A “suicide” gene also can be incorporatedinto a vector so as to allow for selective inducible killing of a cell,particularly of a genetically modified tumor cell, when the treatment iscompleted or otherwise terminated. A gene such as the herpes simplexvirus thymidine kinase gene (TK) can be used as a suicide gene toprovide a means of inducible destruction of a cell. For example, when acell such as a tumor cell no longer is useful in the subject, a drugsuch as acyclovir or gancyclovir can be administered to the subject.Either of these drugs selectively kills cells expressing a viral TK,thus eliminating the genetically modified cells. Additionally, a suicidegene can encode a non-secreted cytotoxic polypeptide and can be linkedto an inducible promoter. If destruction of the cells is desired, theappropriate inducer of the promotor is administered so that thecytotoxic polypeptide is expressed.

[0061] Numerous methods are available for transferring nucleic acidsequences into cultured cells, including the methods described above. Inaddition, a useful method can be similar to that employed in previoushuman gene transfer studies, where tumor infiltrating lymphocytes (TILs)were modified by retroviral gene transduction and administered to cancersubjects (Rosenberg et al., New Engl. J. Med. 323:570-578 (1990); see,also, U.S. Pat. No.: 5,460,959, issued Oct. 24, 1995; U.S. Pat. No.5,399,346, issued Mar 21, 1995; each of which is incorporated herein byreference). In the Phase I safety study of retroviral mediated genetransfer, TILs were genetically modified to express the neomycinresistance gene. Following intravenous infusion, polymerase chainreaction analyses consistently found genetically modified cells in thecirculation for as long as two months after administration. Noinfectious retroviruses were identified in these subjects and no sideeffects due to gene transfer were noted in any subjects. Theseretroviral vectors have been altered to prevent viral replication by thedeletion of viral gag, pol and env genes.

[0062] When retroviruses are used for gene transfer, replicationcompetent retroviruses theoretically can develop due to recombination ofretroviral vector and viral gene sequences in the packaging cell lineutilized to produce the retroviral vector. Packaging cell lines in whichthe production of replication competent virus by recombination has beenreduced or eliminated can be used to minimize the likelihood that areplication competent retrovirus will be produced. Hence, all retroviralvector supernatants used to infect subject cells will be screened forreplication competent virus by standard assays such as PCR and reversetranscriptase assays.

[0063] As discussed above, a cancer cell can express animmunosuppressive agent such as an immunosuppressive isoform of TGFβ.Such cancer cells should. be genetically modified to reduce or inhibitthe expression of the immunosuppressive agent if the cells are to beused as carrier cells or are to be administered in combination with animmunostimulatory agent such as a cytokine, CEcells or the like.Reduction or inhibition of expression of an immunosuppressive agent thatis expressed by a tumor cell can be accomplished using known methods ofgenetic modification. For example, a tumor cell expressing animmunosuppressive agent such as an immunosuppressive isoform of TGFβcanbe genetically modified such that the expression of the TGFβ is reducedor inhibited using a homologous recombination gene “knock-out” method(see, for example, Capecchi, Nature, 344:105 (1990) and references citedtherein; Koller et al., Science, 248:1227-1230 (1990); Zijlstra et al.,Nature, 342:435-438 (1989), each of which is incorporated herein byreference; see, also, Sena and Zarling, Nat. Genet., 3:365-372 (.1993),which is incorporated herein by reference). The homologous recombinationgene knock-out method provides several advantages. For example,expression of a gene encoding an immunosuppressive agent such as a TGFβgene in a tumor cell can be inhibited completely if both alleles of thetarget gene are inactivated. In addition to providing completeinhibition of the immunosuppressive agent, the method of homologousrecombination gene knock-out is essentially permanent.

[0064] The expression of an immunosuppressive agent by a tumor cell alsocan be reduced or inhibited by providing in the tumor cell an antisensenucleic acid sequence, which is complementary to a nucleic acid sequenceor a portion of a nucleic acid sequence encoding an immunosuppressiveagent such as an immunosuppressive isoform of TGFβ. Methods for using anantisense nucleic acid sequence to inhibit the expression of a nucleicacid sequence are known in the art and described, for example, by Godsonet al., J. Biol. Chem., 268:11946-11950 (1993), which is incorporatedherein by reference. Expression of an immunosuppressive agent by a tumorcell also can be reduced or inhibited by providing in the tumor cell anucleic acid sequence encoding a ribozyme, which can be designed torecognize and inactivate a specific mRNA such as a mRNA encoding animmunosuppressive isoform of TGFβ (see, for example, McCall et al.,Proc. Natl. Acad. Sci., USA, 89:5710-5714 (1992); Cremisi et al., Proc.Natl. Acad. Sci., USA, 89:1651-1655 (1992); Williams et al., Proc. Natl.Acad. Sci., USA, 89:918-921 (1992); Neckers and Whitesell, Amer. J.Physiol. 265:L1-12 (1993); Tropsha et al., J. Mol. Recog. 5:43-54(1992), each of which is incorporated herein by reference).

[0065] Various assays to determine whether a subject's cancer cellsexpress an immunosuppressive agent such as an immunosuppressive isoformof TGFβ are available and known to those skilled in the art. Forexample, a radioimmunoassay or enzyme linked immunosorbent assay can beused to detect a specific immunosuppressive agent in a serum or urinesample obtained from a subject. In addition, an assay such as the minklung epithelial cell assay can be used, for example, to identify TGFβ2activity (ogawa and Seyedin, Meth. Enzymol. 198:317-327 (1991), which isincorporated herein by reference). A biopsy of the tumor also can beexamined, for example, immunohistochemically for the expression of animmunosuppressive agent. In addition, the tumor cells can be evaluatedby northern blot analysis, reverse transcriptase-polymerase chainreaction or other known methods (see, for example, Erlich, PCRTechnology: Principles and applications for DNA amplification (StocktonPress 1989); Sambrook et al., Molecular Cloning: a laboratory manual(Cold Spring Harbor Laboratory Press 1989), each of which isincorporated herein by reference).

[0066] It is recognized that, in order to sensitize a subject's cancerto radiotherapy, the cytbkine must be expressed in an effective amount.As used herein, the term “effective amount” means an amount of acytokine that can stimulate a systemic immune response in the subject oralter the tumor microenvironment, such that a tumor in the subject isradiosensitized. An effective amount can be determined, for example, bythe detecting a stimulation of the subject's immune response or bydetecting a reduction in the severity of the subject's cancer followingradiotherapy. Such an effective amount can be determined using assaysfor determining the activity of immunoeffector cells followingadministration of an immunizing composition to the subject or bymonitoring the effectiveness of the radiotherapy using well knownimaging methods.

[0067] Where an immunizing composition includes a genetically modifiedcell such as a tumor cell, carrier cell or a CE cell, the number ofcells to be administered depends, in part, on the amount of cytokinesecreted by the cells. Methods for determining the level of a cytokineexpressed by a genetically modified cell are disclosed herein orotherwise known in the art (see, for example, Thomson, supra, 1994;Chap. 25). For example, the IL-3 expressing cells generated as describedin Example I produced 50 ng bioactive IL-3/ml medium/48 hr/1×10⁶ cells.In general, about 1×10⁵ to about 1×10⁷ cells is required forimmunization, depending, for example, on the number of times thecomposition is to be administered, as well as the amount of a particularcytokine secreted.

[0068] Prior to administration, genetically modified cells can be mixedwith an appropriate adjuvant or with a pharmacologically acceptablesolution such as physiological saline or the like for administration,which can be accomplished by any of various methods such as subcutaneousor intramuscular injection or any manner acceptable for immunization.Pharmacologically acceptable solutions useful for administration to asubject are known in the art (see, for example, Khan et al., supra,1994; Audibert and Lise, supra, 1993; Mishell and Shiigi, supra, 1980).In addition, various methods of administration can be used and are knownto those skilled in the art. Administration can be at a body locationother than an active tumor site or, if desired, at the site of a tumorin a cancer subject.

[0069] One skilled in the art would know that the effectiveness oftherapy can be determined by monitoring a subject'simmunoresponsiveness. For example, the cytolytic activity of immuneeffector cells against the subject's cancer cells can be assayed usingwell known methods. In addition, the size or growth rate of a tumor canbe monitored in vivo using methods of diagnostic imaging.

[0070] It is understood that modifications that do not substantiallyaffect the embodiments of this invention also are included within theinvention provided herein. Accordingly, the following examples areintended to illustrate but not limit the present invention.

EXAMPLE I Expression of IL-3 at the Site of a Tumor Radiosensitizes theTumor

[0071] This example demonstrates that a tumor consisting of tumor cellsgenetically modified to express IL-3 is more radiosensitive than a tumorconsisting of unmodified tumor cells.

[0072] A. IL-3 Transduction:

[0073] The Jzen.1 retroviral vector (Laker et al., Proc. Natl. Acad.Sci., USA 84:8458-8462 (1987), which is incorporated herein byreference) was used to introduce the full length cDNA encoding murineIL-3 into cultured non-immunogenic (FSAN) or moderately immunogenic(FSA) murine fibrosarcoma cells as previously described (McBride et al.,supra, 1992; McBride et al., supra, 1994; see, also, McBride and Howie,Br. J. Canc. 53:707-711 (1986), which is incorporated herein byreference).

[0074] Briefly, the IL-3 cDNA was inserted downstream of the 5′-LTRpromoter and upstream of a neo gene driven by an internal TK promotorpresent in the vector. The IL-3-containing vector was transfected usingcalcium phosphate precipitation into the ecotropic packaging cell lineGP+env−86 (Markowitz et al., J. Virol. 62:1120-1124 (1988), which isincorporated herein by reference). Cloned cell lines produced viraltiters in excess of 1×10⁶ pfu/ml.

[0075] FSA and FSAN tumor cells were infected with the IL-3-containingvector (Jzen-IL-3) or with the parental vector lacking the IL-3 cDNAinsert (Jzen) and infected cells were selected in 0.6 mg G418/ml medium.Sequential daily supernatant infections with the retroviral vectors over3 days transduced about 50% of the cells.

[0076] Expression of IL-3 mRNA by the Jzen-IL-3 transfected cell lineswas confirmed by northern blot analysis using the murine IL-3 cDNA as aprobe. The IL-3 transduced cells expressed the appropriate 4 kb RNA. Inaddition, IL-3 protein expression was confirmed by ³H-thymidineincorporation into the IL-3-dependent B6SUtA murine mast cell line(McBride et al., supra, 1994). IL-3 transduced cells producedapproximately 50 ng bioactive IL-3/ml medium/1×10⁶ cells in a 48 hrperiod.

[0077] B. IL-3 Expressing Tumor Cells are Immunogenic:

[0078] Subcutaneous injection of (2.5-3.0)×10³ FSA or FSAN cells resultin 50% incidence of tumors (TD-50) in the C3Hf/Sed/Kam female mice(10-12 weeks old) used in these studies. In comparison, IL-3 transducedFSA (FSA-IL-3) or FSAN (FSAN-IL-3) cells had a TD-50 of 1.2×10⁶ or2.8×10⁵ cells, respectively. The decreased tumorigenicity is correlatedwith granulocyte infiltration into the tumors (see McBride et al.,supra, 1994). For all experiments described herein, appropriate controlswere run in parallel, including the comparison of genetically modifiedcells with the corresponding unmodified cells or cells modified with thecontrol Jzen.1 vector.

[0079] The ability of irradiated IL-3 expressing tumor cells to induceimmunity to a subsequent challenge of unmodified tumor cells wasexamined. Cells were irradiated using a Gammacell 220 (Atomic EnergyLimited; Canada) with a cobalt source at a dose rate of 3.3 Gy/min to atotal of 30 Gy. 1×10⁶ irradiated (30 Gy) IL-3 transduced FSA (FSA-IL-3)or FSAN (FSAN-IL-3) cells were injected intraperitoneally into mice,then, 10 days later, the mice were challenged with 1×10⁶ unmodified FSAor FSAN cells, respectively. The number of immunizing IL-3 transducedtumor cells was deliberately chosen to be too low to protect againstgrowth of the moderately immunogenic FSA tumor cells in order allow thedetection of enhanced immunogenicity.

[0080] Immunization with the irradiated IL-3 transduced tumor cells, butnot with Jzen.1 modified tumor cells, induced specific immunity againstthe immunizing cell type (i.e., FSA or FSAN), protecting the mice fromsubsequent challenge with unmodified tumor cells (McBride et al., supra,1994). Complete protection occurred in 80% to 90% of the immunized miceand no cross-protection was observed against the antigenically unrelatedtumor.

[0081] The generation of immunologic memory in the immunized mice wasdemonstrated by intravenously injecting 2×10 ⁷ spleen cells fromimmunized mice or control (not previously treated) mice into syngeneicC3H SCID mice bearing previously established 4 day old parental FSA orFSAN tumors. Regression of the established tumors was observed followingadoptive transfer of spleen cells from the immunized mice but not fromthe control mice. In addition, a moderate amount of cross-protection wasobserved against the non-immunizing tumor cells (McBride et al., supra,1994). These results demonstrate that a specific, systemic immuneresponse is induced in mice immunized with IL-3 expressing tumor cells.

[0082] C. IL-3 Expressing Tumor Cells are Radiosensitized:

[0083] (1-5)×10 ⁶ IL-3 transduced FSA or FSAN cells or Jnez.1 transducedFSA or FSAN cells were injected subcutaneously into the thigh muscles ofmice (N=5-10). After about 16 to 20 days, when the tumor were about 6 to8 mm in diameter, the tumors were irradiated. For irradiation, mice wereplaced in a full body shield, with the tumor bearing thigh exposed.Tumors were irradiated using a 250 kVp Phillip's X-ray source at a doserate of 11.2 Gy/min. The tumors in mice bearing the moderatelyimmunogenic FSA tumors were irradiated with a single dose of 25 Gy or 40Gy. The tumors in mice bearing the non-immunogenic FSAN tumors wereirradiated with a single dose of 55 Gy.

[0084] IL-3 expressing FSA tumors regressed completely followingradiotherapy with 25 Gy or 40 Gy. In comparison, unirradiated IL-3expressing FSA tumors and irradiated unmodified parental FSA tumorscontinued growing during the course of the experiment, although growthof the irradiated, unmodified FSA tumors was slightly delayed followingirradiation. Furthermore, mice that were cured of the IL-3 expressingFSA tumors were protected from challenge with 2×10⁶ unmodified FSA tumorcells, but not of FSAN tumor cells.

[0085] Similarly, IL-3 expressing FSAN tumors regressed completelyfollowing radiotherapy with 55 Gy, whereas Jzen.1 modified FSAN tumorscontinued to grow after a brief delay due to the irradiation. Inaddition, mice cured of the IL-3 expressing FSAN tumors were protectedfrom challenge with unmodified FSAN cells.

[0086] These results demonstrate that the expression of IL-3 at the siteof tumor radiosensitizes the tumor, thus allowing complete regression ofan otherwise progressive tumor. In addition, a specific, systemic immuneresponse was generated such that no tumors formed upon further challengewith tumor cells.

EXAMPLE II General Considerations for Treatment of a Subject

[0087] This example illustrates the general factors to consider intreating a cancer patient with a method of the invention.

[0088] Patient Selection:

[0089] Patients will have a histologically confirmed diagnosis of cancerand can have metastatic lesions. Patients with tumors that must beresected for therapeutic purposes or with tumors readily accessible forbiopsy can be treated as disclosed herein. Autologous fibroblasts andtumor cells can be cultured using routine methods. However, where theautologous tumor cells are not amenable to growth in culture, tumorantigens can be provided by sources as disclosed herein. Thus,allogeneic haplotype-matched genetically modified tumor cells can beused provided such tumor cells are of the same histologic origin as thepatient's tumor.

[0090] Pretreatment Evaluation:

[0091] Standard pretreatment evaluations are performed as follows:

[0092] 1) History and physical examination including a description andquantitation of disease activity and tissue-typing of the patient.

[0093] 2) Performance Status Assessment

[0094] 0=Normal, no symptoms

[0095] 1=Restricted, but ambulatory

[0096] 2=Up greater than 50% of waking hours, capable of self-care

[0097] 3=Greater than 50% of waking hours confined to bed or chair,limited self-care

[0098] 4=Bedridden

[0099]3) Pretreatment laboratory analysis, including complete bloodcount, including differential count, platelet count, PT, PTT, glucose,BUN, creatinine, electrolytes, SGOT, SGPT, LDH, alkaline phosphatase,bilirubin, uric acid, calcium and total protein albumin.

[0100] Other analyses are performed as deemed appropriate, includingurinalysis, serum complement levels and immunophenotyping of peripheralblood B cell and T cell subsets. In addition, pretreatment evaluationscan include chest X-ray and other diagnostic studies includingcomputerized tomography, magnetic resonance imaging or radionuclidescans to document and quantify the extent of disease activity. Follow-upevaluations of these assessments are performed at regular intervalsduring the course of therapy (approximately every 1 to 3 months) tomonitor the subject's response to therapy and to identify potentialsigns of toxicity, thus permitting adjustments in the number anddistribution of immunizations.

[0101] Restrictions on Concurrent Therapy:

[0102] For optimal effects of this treatment, patients should receive noconcurrent therapy which is known to suppress the immune system.

[0103] Treatment Protocol:

[0104] Each patient will receive either intratumoral or subcutaneousadministrations an immunizing composition, which can be provided in theform of autologous or allogeneic haplotype-matched carrier cells or inthe form of CE cells and a tumor antigen, if desired. Tumor cellsgenerally will be irradiated. Prior to administration, tumor cells canbe irradiated with approximately 70 to 100 Gy of radiation, to renderthe tumor cells incapable of proliferation in vivo.

[0105] When an immunizing composition is administered to the site of atumor, radiotherapy can begin one to three days following theadministration. When immunization is at a site other than the tumor,immunization generally will require two to four administrations of theimmunizing composition at one to four week intervals, with adjustmentsbeing made as required. Radiotherapy can begin concurrently with theimmunization protocol, since the localized radiation will notsubstantially affect the immunoresponsiveness of the subject.Preferably, radiotherapy will begin after it has been determined thatthe patient's immune response has been stimulated by the immunizations.

[0106] Conventional methods of radiotherapy are performed. Thestimulation of a patient's immune response will be determined bystandard methods, including, for example, detecting the presence ofactivated immunoeffector cells either in vitro or by detecting a delayedhypersensitivity-type reaction.

[0107] In general, a tumor biopsy is taken approximately two monthsprior to the initiation of the methods disclosed herein. If the tumorcells are adaptable to tissue culture, they can be genetically modifiedto express a cytokine gene and used as carrier cells. However, even ifthe tumor cells cannot be grown in culture, they can be stored underappropriate conditions and used as a source of tumor antigen, ifdesired.

[0108] The immunizing composition is administered in a form thatprovides controlled slow release of the cytokine, particularly whenadministration is at a site other than the tumor. In addition, if atumor cell or a carrier cell is not the source of the cytokine,irradiated unmodified tumor cells also can be administered as a sourceof tumor antigen, particularly when administration is at a site otherthan the tumor.

[0109] Where administration involves, for example, the use ofIL-3-expressing cells, the level of IL-3 secreted at the site ofimmunization can be escalated as required during the immunizationprocedure. The number of injected IL-3-expressing cells will remainrelatively constant at approximately 1×10⁵ to 1×10⁷ cells peradministration site by adding an appropriate number of irradiatedunmodified cells. Multiple immunization sites can be used if it isdeemed desirable to increase the dose of the cytokine to the subject.The subject will be physically examined on each of the three consecutivedays following administration and physical and laboratory evaluationswill be made at weekly intervals.

[0110] Dose Adjustments:

[0111] The immunoresponsiveness of the subject is determined using theassays described above, including, for example, assays to determinechanges in the activity of the cellular immune response in the subject.So long as no toxicity is observed, subsequent administrations areadministered at intervals of 1 to 4 weeks, as desired. The results ofthe cellular and humoral immunoresponsiveness assays and tumormonitoring studies can be used to optimize the treatment protocol asdetermined by one skilled in the art. Although toxic side effects arenot expected to result, potential side effects can be treated usingconventional methods.

[0112] Treatment of Potential Toxicity:

[0113] Unacceptable toxic side effects at the site of administration arenot expected to result during the course of treatment. However,potential side effects can be treated as required. For example, ifmassive tumor cell lysis results, any resulting uric acid nephropathy,adult respiratory distress syndrome, disseminated intravascularcoagulation or hyperkalemia will be treated using standard methods wellknown in the art. Local toxicity at the sites of administration will betreated with either topical steroids and, if necessary, surgicalexcision of the injection site. Generalized hypersensitivity reactionssuch as “the chills,” fever or rash will be treated symptomatically withantipyretics and antihistamines. Patients should not be treatedprophylactically. Edema, arthralgia, lymphadenopathy or renaldysfunction can be treated using corticosteroids and/or antihistamines.Anaphylaxis will be treated by standard means such as administration ofepinephrine, fluids and steroids.

[0114] Other Assays:

[0115] Provided that sufficient material is available for evaluation,the following assays also are performed. Standard immunofluorescenceflow cytometry procedures are useful to evaluate changes in thepercentage of T cells, natural killer cells and B cells associated withcytokine gene therapy. Monoclonal antibodies specific for T cells (CD2,CD3, CD4, CD8), natural killer cells (CD16, CD57, CD58) and B cells(CD19, CD20) can be used for these studies.

[0116] Briefly, Ficoll-Hypaque purified mononuclear cells are incubatedwith the primary antibody for 1 hr at room temperature, washed, thenincubated with fluorochrome conjugated secondary antibody. The cells arewashed, fixed and the percentage of positive cells are determined usinga Coulter Epics 4 flow cytometer. Incubation of the cells withisotype-matched control antibody instead of the primary antibody isuseful as a negative substitution control.

[0117] Standard immunohistological methods employing monoclonalantibodies specific for the hematopoietic cell subsets described abovecan be used to characterize the immune effector cell infiltratesobserved in delayed-type hypersensitivity type skin test biopsy sites.Methods for immunohistological evaluations of fresh frozen cryostattissue sections are well known in the art.

[0118] Although the invention has been described with reference to theabove examples, it is understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

I claim:
 1. A method of radiosensitizing a tumor in a subject,comprising contacting the tumor with a cytokine, provided that saidcytokine is not interleukin-7 or tumor necrosis factor-α.
 2. The methodof claim 1, wherein said cytokine is interleukin-3.
 3. The method ofclaim 1, wherein said cytokine is selected from the group consisting ofgranulocyte-macrophage colony stimulating factor and granulocyte colonystimulating factor.
 4. The method of claim 1, wherein said contactingcomprises administering, at the site of said tumor, a cell that has beengenetically modified to express said cytokine.
 5. The method of claim 4,wherein said cell that has been genetically modified is a tumor cell. 6.The method of claim 4, wherein said cytokine is interleukin-3.
 7. Themethod of claim 4, wherein said cytokine is selected from the groupconsisting of granulocyte-macrophage colony stimulating factor andgranulocyte colony stimulating factor.
 8. A method of radiosensitizing atumor in a subject, comprising contacting the tumor with a nucleic acidmolecule encoding a cytokine, provided that said cytokine is notinterleukin-7 or tumor necrosis factor-α.
 9. The method of claim 8,wherein said cytokine is interleukin-3.
 10. The method of claim 8,wherein said cytokine is selected from the group consisting ofgranulocyte-macrophage colony stimulating factor and granulocyte colonystimulating factor.
 11. A method of radiosensitizing a tumor in asubject, comprising administering to the subject, at a site other thanthe tumor, an immunizing composition comprising a cytokine and a tumorantigen.
 12. The method of claim 11, wherein said immunizing compositionis a cell genetically modified to express said cytokine and said tumorantigen.
 13. The method of claim 11, wherein said immunizing compositionis a tumor cell, which expresses said tumor antigen and is geneticallymodified to express said cytokine.
 14. The method of claim 11, whereinsaid cytokine is interleukin-3.
 15. The method of claim 11, wherein saidcytokine is selected from the group consisting of granulocyte-macrophagecolony stimulating factor and granulocyte colony stimulating factor. 16.A method of reducing the severity of a cancer in a subject, comprisingthe steps of: a) immunizing said subject with tumor cells geneticallymodified to express a cytokine; and b) administering a radiotherapeuticdose of radiation to the site of the cancer.
 17. The method of claim 16,wherein said cytokine is interleukin-3.
 18. The method of claim 16,wherein said immunostimulatory agent is selected from the groupconsisting of granulocyte-macrophage colony stimulating factor andgranulocyte colony stimulating factor.